WO2013097657A1 - Arginase humaine et arginase humaine pégylée à point fixe, et leur utilisation - Google Patents

Arginase humaine et arginase humaine pégylée à point fixe, et leur utilisation Download PDF

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Publication number
WO2013097657A1
WO2013097657A1 PCT/CN2012/087240 CN2012087240W WO2013097657A1 WO 2013097657 A1 WO2013097657 A1 WO 2013097657A1 CN 2012087240 W CN2012087240 W CN 2012087240W WO 2013097657 A1 WO2013097657 A1 WO 2013097657A1
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Prior art keywords
arginase
pegylated
cysteine
mutated
amino acid
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English (en)
Chinese (zh)
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郑宁民
陈丽
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BIO-CANCER TREATMENT INTERNATIONAL Ltd (SHANGHAI)
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BIO-CANCER TREATMENT INTERNATIONAL Ltd (SHANGHAI)
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Priority to EP12861903.8A priority Critical patent/EP2799539B1/fr
Priority to US14/368,508 priority patent/US20150010522A1/en
Priority to BR112014015803-7A priority patent/BR112014015803B1/pt
Priority to KR1020147020855A priority patent/KR102076348B1/ko
Priority to JP2014549335A priority patent/JP2015503333A/ja
Publication of WO2013097657A1 publication Critical patent/WO2013097657A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03001—Arginase (3.5.3.1)

Definitions

  • the present invention relates to an arginase, a process for its preparation and its use in the treatment of diseases associated with arginase. Specifically, the present invention provides a point mutation arginase, a preparation method thereof, and use thereof in the treatment of a disease associated with arginase. The present invention also provides a site-specific PEGylated arginase, a preparation method and application thereof. Background technique
  • Arginine is an important amino acid of mammals, including humans, involved in various physiological processes, including cell proliferation and growth.
  • arginine can serve as a direct precursor to the synthesis of a potential signaling molecule, nitrogen oxides (NO).
  • NO acts as a neurotransmitter, a smooth muscle relaxant and a vasodilator.
  • the biosynthesis of NO involves Ca ++ and NADPH-dependent reactions catalyzed by nitric oxide synthase.
  • Another role of arginine is as a precursor to polyamines, spermidine or spermine, involved in different physiological processes.
  • Arginase is an enzyme that catalyzes the hydrolysis of L-arginine to produce ornithine and urea, and is generally contained in animals that produce urea (mammals, squid, two-handed, sea turtles). Liver, kidney, testis, as a urea cycle One link works.
  • Arginase is an enzyme that catalyzes the final step of urea formation in the mammalian urea cycle pathway, converting arginine to ornithine and urea.
  • the human arginase family includes arginase I and arginase II.
  • Arginase I is mainly expressed in liver cells
  • arginase II is mainly expressed in kidney and red blood cells.
  • Arginase can be obtained by two methods, one is isolated from the organism in which it is produced, and the other is obtained by genetic engineering techniques.
  • the superiority of recombinant production of arginase by genetic engineering technology exists. For example, experiments have shown that E. coli can produce large amounts of arginase.
  • recombinant production of arginase by genetic engineering techniques sometimes has technical problems, such as low enzyme activity, poor stability in vivo, and short half-life, which cannot be applied to actual clinical practice.
  • US Pat. No. 7,951,366 B2 discloses a pharmaceutical composition and method for treating human malignancies using arginine deprivation.
  • Recombinant human arginase I with his-tag was used to covalently conjugate a group with an amino group in the N-terminus of the arginase or amino acid in the surface amino acid with a molecular weight of 5,000 (MW 5,000). Coupled with polyethylene glycol modification, the stability of the modified human arginase is increased, and the half-life in human serum is 3 days.
  • US20100247508A1 modified human arginase with his-tag, replacing cysteines 168 and 303 with serine, and using polyethylene glycol with a molecular weight of 20 kDa for human arginase Make modifications.
  • the arginase has an additional polypeptide fragment such as His-tag.
  • Most drug regulatory agencies in most countries do not recommend the use of these peptide fragments.
  • the State Food and Drug Administration of China has proposed in the "Guidelines for Quality Control Technology for Human Recombinant DNA Products" to simplify the production process.
  • the polypeptide fragment, such as His-tag should be removed as much as possible in the final product.
  • WO 2011/008495 A2 discloses the addition of a cysteine residue at the position of the third amino acid at the N-terminus, based on the retention of the three cysteines carried by the human arginase itself.
  • the human arginase to which a cysteine residue is added is then modified with an oxirane polyethylene glycol maleimide having a molecular weight of 20 kDa.
  • the three cysteines carried by the human arginase itself still exist, and the formed PEGylated human arginase product is heterogeneous and has a low yield, which is difficult to separate and purify.
  • the invention provides an isolated and substantially purified arginase.
  • Arginase is an enzyme that catalyzes the final step of urea formation in the mammalian urea cycle pathway, converting arginine to ornithine and urea.
  • the arginase family includes arginase I and arginase II. Sequencing and activity studies of arginase I from various sources have revealed that the sequences of arginase I from different sources may differ, but also have many conserved regions and active sites.
  • wild type human arginase I has the amino acid sequence of SEQ ID NO. 1, with three vesicles therein. The amino acid is located at positions 45, 168 and 303 of the amino acid sequence of SEQ ID NO. 1, respectively, and the gene has the nucleic acid sequence of SEQ ID NO.
  • the term “isolated” refers to a non-natural form.
  • substantially purified means that there may be other ingredients used to produce and/or modify the protein, but the other ingredients are substantially absent or less proportionate throughout the product.
  • the invention provides a mutant arginase, characterized in that the arginase is human arginase I, which has
  • amino acid sequence obtained by substitution, deletion, insertion, addition or inversion of one or several amino acids is also introduced in the above amino acid sequence, and has arginase activity.
  • the cysteine is mutated to a non-polar amino acid in the arginase of the invention.
  • Amino acids can be classified into polar amino acids and non-polar amino acids depending on the nature of the side chain groups.
  • the side chain group of an amino acid is uncharged or has a very weak polarity, such as: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, color Amino acid, valine.
  • the cysteine mutation in the arginase of the invention is glycine, alanine, valine, leucine, isoleucine, methionine, styrene Amino acid, tryptophan or valine.
  • the cysteine is mutated to alanine in the arginase of the present invention.
  • a cysteine belonging to a non-ionized polar amino acid in a human arginase I sequence is mutated to an amino acid belonging to a non-polar amino acid property (for example, alanine), and the enzyme The activity is significantly increased.
  • a non-polar amino acid property for example, alanine
  • the arginase of the invention is at position 45 of the amino acid sequence of SEQ ID NO.
  • the refined ammonia of the invention is mutated to a cysteine at position 303 of the amino acid sequence of SEQ ID NO. 1, which is preferably mutated to glycine, alanine, valine, leucine, isoleucine, More preferably, methionine, phenylalanine, tryptophan or valine is mutated to alanine.
  • the arginase of the invention is mutated in any two of the cysteines at positions 45, 168 and 303 of the arginase.
  • the arginase of the invention is mutated at positions 45 and 303 of the amino acid sequence of SEQ ID NO. 1, preferably mutated to glycine, Alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, more preferably, mutated to alanine.
  • the arginase of the invention is mutated to a cysteine at positions 168 and 303 of the amino acid sequence of SEQ ID NO.
  • the arginase of the invention is mutated at positions 45 and 168 of the amino acid sequence of SEQ ID NO. 1, the cysteine preferably being mutated to glycine, Alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, more preferably, mutated to alanine.
  • the arginase of the invention is mutated at positions 45, 168 and 303 of the amino acid sequence of SEQ ID NO. 1, said cysteine
  • the amino acid is mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, and more preferably, mutated to alanine.
  • the arginase of the present invention has a stronger activity than wild-type arginase.
  • the specific activity of the arginase of the present invention is generally at least about 500 U/mg, preferably at least about 700 U/mg, and most preferably at least about 800 U/mg.
  • the arginase of the present invention generally has a stronger activity than the wild type arginase.
  • the arginase of the invention has the polynucleotide of the base sequence of SEQ ID NO. 2, and wherein at least one of the codes corresponds to amino acid sequence 45, 168 of SEQ ID NO.
  • the base sequence of the cysteine at position 303 was replaced.
  • one, two or three base sequences encoding cysteine are substituted.
  • Amino acids are encoded by polynucleotides. Messengers in a polynucleotide The three bases on the R A chain that determine an amino acid are called a "codon", also known as a triplet code.
  • the substitution of the base is replaced by TGT to encode glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, guanidine
  • the base sequence of the acid preferably, is replaced by a base sequence encoding alanine.
  • the base sequence encoding alanine is 0 « ⁇ 00,0[,0[0, preferably For GCT.
  • the invention provides a method of making the above arginase comprising expressing an arginase gene or a mutated arginase gene in a host in which the protein can be expressed.
  • the method of preparing an arginase of the present invention generally comprises the following main steps.
  • the gene of interest or the nucleotide fragment of interest is obtained by PCR or synthetic methods.
  • a DNA fragment carrying the gene of interest is ligated to a vector capable of independent replication and having a selectable marker, such as a plasmid, a bacteriophage, and a virus, to form a recombinant DNA molecule.
  • the DNA fragments are ligated to the vector mainly by homopolymeric ends, sticky end connections, flush end connections, and artificial linker molecules.
  • Recombinant DNA must enter the host cell for amplification and expression.
  • recombinant DNA molecules can be introduced into host cells by means of transfection, transformation, transduction, etc., and allowed to multiply.
  • Suitable genetically engineered hosts are well known in the art and may be E. coli, yeast, insect cells, and the like.
  • the invention also provides an isolated and substantially purified PEGylation arginase, characterized in that the pegylation is site specific.
  • the polyethylene glycol molecule is covalently bound to a specific amino acid residue on the arginase molecule to achieve site-specific pegylation.
  • each arginase molecule binds to at least one polyethylene glycol molecule to form a PEGylated arginase.
  • the arginase enzyme of the PEGylated arginase of the invention is a mutant arginase as described above.
  • the invention provides a PEGylated arginase, wherein the arginase is human arginase I, which has
  • amino acid sequence obtained by substitution, deletion, insertion, addition or inversion of one or several amino acids is also introduced in the above amino acid sequence, and has arginase activity.
  • the cysteine point mutation in the PEGylated arginase of the invention is a non-polar amino acid.
  • the structural differences in the 20 protein amino acids depend on the difference in side chain groups.
  • the side chain group of an amino acid is uncharged or has a very weak polarity, such as: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, color Amino acid, valine.
  • the sequence of the pegylated human arginase I does not have a tag sequence for purifying the protein, for example, a His-tag sequence is added at its C-terminus or N-terminus.
  • His-Tag fusion protein is currently the most A common expression, which has the advantage of being convenient to purify and does not substantially affect the activity of the protein, whether the expressed protein is soluble or the inclusion body can be purified by immobilized metal ion affinity chromatography.
  • the State Food and Drug Administration of China has proposed in the "Guidelines for Quality Control Techniques for Human Recombinant DNA Products" to introduce additional peptide fragments such as His-tags for the purpose of simplifying the production process. It should be removed as much as possible in the final product.
  • pegylation of arginase can be achieved by a chemical modification method which can be carried out by a polyethylene glycol derivative (also referred to as polyethylation) with a coupling agent. a diol modifier) that covalently binds a polyethylene glycol molecule to a group on the arginase.
  • the chemical modification may be specific, i.e., by using a coupling agent that binds to a particular group, the PEG can only bind to a particular amino acid on the arginase.
  • Site-specific PEGylation modification should follow the following principles: (1) Try to avoid modification of the active site of the protein to prevent the activity of the protein; (2) Simplify the whole process as much as possible, which is controllable.
  • a common polyethylene glycol molecule has a hydroxyl group at each end, and if one end is blocked with a thiol group, a decyloxy polyethylene glycol (mPEG) is obtained.
  • mPEG decyloxy polyethylene glycol
  • the most studied polyethylene glycol modifiers in the polyethylene glycol modification of polypeptides and proteins are derivatives of mPEG.
  • One of the common methods of modifying polyethylene glycol is to combine a polyethylene glycol molecule/polyethylene glycol modifier with lysine on the surface of the protein or ⁇ - ⁇ 2 or ⁇ - ⁇ 2 at the N-terminus of the protein, such as mPEG-succinyl.
  • Modifiers such as mPEG-maleimide, mPEG-o-pyridine-disulfide, mPEG-vinyl sulfone, mPEG-iodoacetamide, etc., to modify the sulfhydryl group of a protein or polypeptide.
  • the sparse base is relatively small, and it is easier to form a homogenous product.
  • the human arginase I monomer has three cysteine residues located at positions 45, 168 and 303 of the amino acid sequence, respectively.
  • the PEGylated arginase is site-specifically PEGylated at one or both of the cysteine sites at positions 45, 168 and 303.
  • the site-directed pegylation is achieved by genetic engineering site-directed mutagenesis of the arginine cysteine followed by cysteine-specific PEGylation.
  • the cysteine-specific PEGylation modification is by using a decyloxy polyethylene glycol maleimide coupling agent to occlude PEG with arginase The S group of the acid is covalently bound.
  • one or two cysteine mutations that do not require PEGylation in the arginase of the invention are preferably mutated to glycine, alanine, valine, leucine Acid, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably mutated to alanine.
  • the choice of PEG molecular weight takes into account both biological activity and pharmacokinetic factors. Previous studies have found that the time of action of the modified protein drug in the body is related to the amount and molecular weight of the coupled PEG.
  • the PEG used in the present invention may have a molecular weight in the range of 5K to 40K Daltons, may be linear or branched, and may be a PEG derivative as described in the art.
  • the PEG molecules covalently bound to the arginase molecule in the present invention are not limited to a specific type. In one aspect of the invention, the PEG molecule PEGylated to arginase has an average molecular weight of 20K, 30 or 40K.
  • the PEGylated protein enters the body by intravenous injection, and the average molecular weight of the polyethylene glycol affects its half-life in serum.
  • glomeruli can filter protein molecules with molecular weights less than 70 kDa; for PEG, glomeruli can filter PEG molecules with molecular weights less than 30 kDa.
  • the molecular weight of PEG is generally unpredictable for the half-life and activity of the protein to be bound, and is closely related to the molecular weight of the protein, the mode of action, the active site, the PEG binding site, and the like.
  • the PEGylated arginase is site-specifically PEGylated at positions 45 and 168 of the amino acid sequence defined by SEQ ID NO.
  • the site-directed pegylation is achieved by mutating a cysteine at position 303 of the arginase, wherein the PEG has an average molecular weight of 20K or 30K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase is site-specifically PEGylated at position 168 of the amino acid sequence defined by SEQ ID NO. 1, wherein the site-specific PEGylation This was achieved by mutating the 45th and 303th cysteine of the arginase, wherein the PEG has an average molecular weight of 40K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase is site-specifically PEGylated at position 45 of the amino acid sequence defined by SEQ ID NO. 1, wherein the site-specific PEGylation is This was achieved by mutating the 168th and 303th cysteines of arginase, wherein the PEG has an average molecular weight of 40K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase enzymes provided herein generally have a purity in excess of more than 70%, preferably in excess of 80%, and most preferably in 90%.
  • the "purity of PEGylated arginase" as used in the present invention means PEGylated arginase (ie, covalently bonded) in a chemically modified production of PEGylation of arginase
  • the arginase of PEG accounts for the ratio of total arginase (arginase covalently bound to PEG, arginase not covalently bound to PEG and polyPEGylated arginase).
  • the product after PEGylation also contains arginase which is not covalently bound to PEG and polyPEGylated arginase
  • purification of the product is required.
  • Commonly used purification means are well known in the art, including cation exchange columns and the like.
  • the PEGylated arginase provided by the present invention has a half-life in blood or serum of at least 0.5 days, preferably at least 2.5 days, and most preferably at least 3.5 days.
  • the half-life of arginase can be measured by methods well known and commonly used in the art.
  • the measurement of the half-life of arginase in blood or serum is related to the animal model used.
  • the half-life data obtained in animal models with faster metabolic rates (e.g., rats) is generally shorter than data obtained in animal models with faster metabolic rates (e.g., humans).
  • the invention also provides a method of preparing a pegylation arginase.
  • the pegylation is site specific.
  • the polyethylene glycol molecule of the PEGylated arginase of the present invention binds to a specific amino acid residue on the arginase molecule to achieve site-specific pegylation.
  • each arginase molecule binds to at least one polyethylene glycol molecule to form a PEGylated arginine.
  • site-directed pegylation of arginase can be achieved by a chemical modification method in which PEG is covalently covalent with a group on arginase by using a coupling agent. Combine.
  • the chemical modification may be specific, i.e., by using a coupling agent that binds to a particular group, the PEG can only bind to a particular amino acid on the arginase.
  • the present invention also provides the use of any of the above-described arginase or pegylated arginase of the present invention or a pharmaceutical composition thereof for the treatment of a disease associated with arginase /use.
  • the diseases associated with arginase are known in the art to include, for example, conditions/diseases/disorders associated with arginine in mammals. Such conditions/diseases/disorders include: hyperarginemia. Due to the lack of arginase activity, the arginine in the patient cannot be lysed into urea and added to the ornithine metabolic cycle.
  • the arginine content in the blood can be 7-10 times higher than normal, and the arginine in the cerebrospinal fluid and urine. It also increased, and the urinary creatinine output increased.
  • such conditions/diseases/disorders also include arginine-dependent cell proliferation or tumors such as liver cancer, melanoma, breast cancer, small cell lung cancer, prostate cancer, lymphoma and leukemia. .
  • the cells can begin to return to the normal physiological cycle; for proliferating cells or tumors It is said that the lack of arginine causes it to enter the S phase through the 'R' point of the G1 phase of the cell cycle, and soon to apoptosis. Such cells or tumors that are proliferating due to lack of arginine are irreversible. Therefore, scientists began to consider the treatment of cell proliferation or tumor by controlling the level of arginine in the body.
  • the present invention also provides a pharmaceutical composition, the active ingredient of which is the above-described arginase or pegylated arginase of the present invention.
  • the pharmaceutical composition may be formulated in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome or the like, wherein the resulting formulation contains one or more human arginase or PEGylated of the present invention.
  • Arginase is the active ingredient and is mixed with an organic or inorganic carrier or excipient suitable for enteral or parenteral application.
  • adjuvants, stabilizers, thickeners, colorants and perfumes can be used.
  • the active ingredient of one or more isolated and substantially purified human arginase or pegylated arginase enzymes is also included in a sufficient amount of the pharmaceutical formulation to produce the desired desired process, condition or disease. effect.
  • the above pharmaceutical compositions may be formulated into a form suitable for oral administration, for example, tablets, tablets, cough drops, hydrated or oleaginous suspensions, dispersed powders or granules, emulsions, hard or soft capsules, or syrups.
  • Oral formulations can be encapsulated according to techniques known in the art to slow the breakdown and absorption in the gastrointestinal tract, thus providing a sustained action over a longer period of time.
  • the formulation may also be in the form of a sterile injectable solution or suspension.
  • the suspension may be formulated according to known methods using dispersing or wetting agents and suspending agents.
  • the pharmaceutical composition of the present invention may be further prepared in the form of a solid, a liquid, a suspension, a micelle, or a liposome.
  • the formulation of the pharmaceutical composition is in a form suitable for oral or injection.
  • Figure 1 shows the nucleic acid sequence of wild type human arginase I.
  • Figure 2 shows the results of a reductive SDS-polyacrylamide gel electrophoresis assay for human arginase expression.
  • Figure 3 shows a liquid chromatogram of human arginase expression.
  • Figure 4 shows the results of gel electrophoresis detection of wild-type arginase I after reduction and non-reduction treatment.
  • Figure 5 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A303, rhArgl-Al 68/303, and rhArgI-A45/303).
  • Figure 6 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • 6a is rhArgI-A45/303
  • rhArgI-A45/l 68/303 is detected by gel electrophoresis after non-reduction treatment fruit.
  • 6b is rhArgI-A45/468, and rhArgI-A45/l 68/303 is subjected to reduction treatment and gel electrophoresis detection results
  • Figure 7 shows the results of non-reducing SDS-polyacrylamide gel electrophoresis of PEGylated human arginase.
  • Example 1 Construction and expression of human arginase I recombinant plasmid pET30a(+)-rharginase-V without His-tag
  • PCR A nucleic acid fragment was obtained and confirmed by agarose gel electrophoresis.
  • the amplified fragment obtained above and the commercially available pET30a(+) expression vector were separately used in a reaction medium containing restriction endonucleases Ndel and Xhol (purchased from promega). After 1.5 hours of C treatment, the digested fragment was reacted with T4 DNA ligase at 16 ° C overnight. The ligated plasmid was then transformed into competent cells DH5a E. coli cells. Screening was performed on LB nutrient agar plates containing 3 ( ⁇ g/ml kanamycin.
  • a plasmid with the correct insert was obtained by restriction endonuclease analysis, called pET30a(+)-rharginase-V, the plasmid The insert was confirmed by sequencing.
  • the insert contained 969 bases, as shown in Figure 1, and its nucleic acid sequence was SEQ ID No. 2.
  • Example 2 Expression of human arginase I recombinant plasmid pET30a(+)-rharginase-V without His-tag and purification of the target protein
  • a single colony of the transformed cell culture plate was picked and transferred to 25 mL of LB medium.
  • the cell is at 37. C, 150 rpm shaking culture to OD600nm reached 0.6-0.8, and the final concentration was 0.2 mM IPTG to induce expression for 3 hours.
  • SDS-P AGE electrophoresis was performed to detect whether the selected clone had the expression of the target protein.
  • the clones with higher expression were selected to prepare the sweet oil bacteria for preservation as engineering bacteria.
  • E. coli engineering bacteria were cultured in a 15 L fermentor in a fed-batch manner, and when the OD600nm reached 12-13, the expression was induced by adding 0.2 mM IPTG to a final concentration of 3-4 hours. The obtained cells were centrifuged and centrifuged, and the supernatant was taken for further separation and purification.
  • Figure 2 is a result of gel electrophoresis detection of human arginase expression.
  • the samples for each lane are:
  • Figure 3 is a liquid chromatogram of human arginase expression.
  • the purity of the arginase obtained by the above chromatographic method was 90% or more.
  • PCR polymerase chain reaction
  • the polymerase chain reaction was carried out according to the method provided in the kit instructions, and then the template plasmid was digested with restriction endonuclease Dpn1, and competent cells were transformed.
  • the obtained recombinant mutant plasmid was sequenced and confirmed.
  • the sequencing results were successfully mutated to the alanine codon (GCT) clone after selection of the cysteine codon (TGT), and inoculated in LB liquid medium for amplification.
  • the mutant recombinant plasmid was extracted using the Wizard Plus Minipreps kit.
  • the recombinant human arginase I of the mutated human arginase I (rhArgl) sequence substituted with alanine at position 303 is represented by rhArgI-A303; the cysteine at position 168 is replaced with C.
  • the recombinant human arginase I of the amino acid is represented by rhArgI-A168; the recombinant human arginase I substituted with alanine at position 45 is represented by rhArgI-A45; cysteine substitution at positions 168 and 303
  • the recombinant human arginase I which is alanine is represented by rhArgI-A168/303; the recombinant human arginase I substituted for cysteine at positions 45 and 303 to alanine is represented by rhArgI-A45/303;
  • Recombinant human arginase I substituted with 168 cysteine for alanine is represented by rhArgI-A45/168; recombinant human arginase with 45, 168 and 303 cysteine replaced by alanine I denoted as rhArgI-A45/l 68/303; the recombinant mutant plasmid containing the human arginas
  • the transformed cell culture plate was selected for a single colony and transferred to 25 mL of LB medium.
  • the cell is at 37. C, 150 rpm shaking culture to OD600nm reached 0.6-0.8, and the final concentration was 0.2 mM IPTG to induce expression for 3 hours.
  • the selected clones were examined for the expression of the protein of interest by polyacrylamide gel electrophoresis (SDS-PAGE). The clones with higher expression were selected to prepare glycerol bacteria for preservation as engineering bacteria.
  • Example 4 Expression and purification of a plasmid containing a site-directed mutant arginase I (rhArgl)
  • the Escherichia coli engineered bacteria including pET30a(+)-rhArgI-A303, pET30a(+)-rhArgI-Al 68/303, pET30a(+) transformed with the mutant human arginase I (rhArgl) plasmid described above -rhArgI- A45/303 , pET30a(+)-rhArgI- A45/168 , or pET30a(+)-rhArgI- A45/168/303 ) was cultured in a 15L fermentor in batches, when the OD600nm reached 12 At -13 hours, expression was induced to a final concentration of 0.2 mM IPTG for 3-4 hours. The obtained cells were centrifuged and centrifuged, and the supernatant was taken for further separation and purification.
  • the collected samples were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and high pressure liquid chromatography to analyze the purity.
  • the purity of the arginase obtained by the above chromatography method is 90% or more.
  • the activity of arginase was determined by measuring the absorbance value of NADPH coupled to urease and glutamate dehydrogenase.
  • the arginase activity unit is defined as: 1 unit (U) of arginase can be at 30. C, pH 8.3 release ⁇ ⁇ urea.
  • the human arginase I (rhArgI) sequence is located at 168/303, 45/303, 45/168 and 45/168/303.
  • the cysteine belonging to the non-ionized polar amino acid is mutated to be non-
  • the post-alanine enzymatic activity of polar amino acid properties is significantly increased.
  • Example 6 SDS-PAGE electrophoresis analysis of wild type and site-directed mutant arginase I (rhArgl)
  • FIG. 4 is a result of gel electrophoresis detection of wild-type arginase I after reduction and non-reduction treatment.
  • the samples in each lane are: 1 , 3 : bacteriostatic supernatant; 2. purified His-tag-free human arginase 1 (non-reducing treatment); M: protein molecular weight standard; 4. purified His-tag-free human arginase I (reduced treatment).
  • Figure 5 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A303, rhArgl-Al 68/303, and rhArgI-A45/303).
  • the samples in each lane are: 1. Arginase I mutant rhArgI-A303 (non-reducing treatment); 2. Arginase I mutant rhArgI-A168/303 (non-reducing treatment); 3. Fine Lysin I mutant rhArgI-45/A303 (non-reducing treatment); M: protein molecular weight standard; 4. arginase I mutant rhArgI-A303 (reduced treatment); 5.
  • FIG. 6a shows the results of gel electrophoresis after purification of the purified arginase I mutant (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • the samples in each lane are: M: protein molecular weight standard; 1. arginase I mutant rhArgI-A45/l 68/303; 2. arginase I mutant rhArgI-A45/168
  • Figure 6b shows the results of gel electrophoresis after purification of the purified arginase I mutants (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • the samples in each lane are: M: protein molecular weight standard; 1. arginase I mutant rhArgI-A45/l 68/303; 2. arginase I mutant rhArgI-A45/168
  • Example 7 Site-directed PEGylation of mutant arginase I (rhArgl) and purification of PEGylated protein
  • the above-mentioned site-modified PEGylated human arginase I was isolated and purified by using a cation exchange column of Macrocap SP to remove residual PEG and a small amount of unreacted protein.
  • the equilibration solution was a phosphate buffer, and the eluent was a phosphate buffer containing 1 M NaCl.
  • the modified protein sample is first diluted with purified water so that the conductance of the sample is the same as that of the equilibrium solution, and the diluted sample is used as a sample for loading. After equilibrating the column with 5 times column volume with an equilibration solution, the sample was loaded.
  • a site-directed mutagenized arginase I (rhArgI-A303) was reacted with a decyloxy polyethylene glycol maleimide (mPEG-MAL-30K) and purified by a Macrocap SP cation exchange column.
  • Site-directed PEGylation of human arginase I wherein the human arginase (presented by A303-M30K(2)) with two molecular weight 30K oxime ethoxylate maleimides was formed. That is, the ratio of all the cysteine groups at positions 45 and 168 to the decyloxy polyethylene glycol maleimide accounts for more than 70% of the total reaction product.
  • a site-directed mutagenized arginase I (rhArgI-A168/303) was reacted with a Y-type polyethylene glycol maleimide (Y-MAL-40K) via a Macrocap SP cation exchange column.
  • Y-MAL-40K Y-type polyethylene glycol maleimide
  • Purified fixed-point PEGylated human arginase I wherein the human arginase (with A168/303-Y40K) with a molecular weight of 40K Y-type polyethylene glycol maleimide was formed.
  • the ratio of the 45-position cysteine group coupled to the decyloxy polyethylene glycol maleimide accounts for more than 85% of the total reaction product.
  • the activity of arginase was determined by measuring the absorbance value of NADPH coupled with urease and glutamate dehydrogenase by the foregoing method, and the fixed-point PEG was calculated at various molecular weights and at different sites.
  • the enzymatic activities of the modified human arginase I A303-M20K (2), A303-M30K (2), A168/303-Y40K and A45/303-Y40K.
  • the results showed that the above-mentioned site-directed PEGylation-modified human arginase I retained arginase activity, and its activity was more than 400 U/mg, ranging from 400-800 U/mg.
  • Example 7 In vivo pharmacokinetic detection of site-directed PEGylated human arginase I
  • Serum arginase I was detected using a double antibody sandwich £1 ⁇ 18 method (the kit was purchased from Shanghai Yikesai Biological Products Co., Ltd.).
  • the anti-human arginase I monoclonal antibody was coated on the ELISA plate, and the sample and the different concentration standards were added to the wells ( ⁇ /well), and the reaction wells were sealed with sealing paper, 37.
  • C incubation for 90 minutes, human arginase I in the sample or standard will bind to the monoclonal antibody to form an immune complex; wash the plate 5 times; add rabbit anti-human arginase I polyclonal antibody ( ⁇ / well), Seal the reaction well with a sealing tape, 37.
  • the present invention modifies the 45, 168 and 303 cysteines in the human arginine sequence by site-directed mutagenesis, and one, two or three codons encoding a cysteine (TGT)
  • TGT cysteine
  • GCT codon
  • the present invention provides a novel site-directed PEGylated human arginase that removes potentially potentially immunogenic his-tag protein tag sequences and ranks 45, 168 and 303 in the human arginine sequence.
  • the polyethylene glycol maleimide was subjected to a coupling reaction.
  • the site-specific PEGylated human arginase synthesized by this method reduces the glomerular filtration rate, and the position of PEG coupled with human arginase is determined, and the formed product is uniform, easy to purify, and is favorable for large scale. Quality control of production.
  • the fixed-point PEGylated human arginase provided by the present invention has a long half-life in mammals, for example, a half-life in serum of rats can reach 15-28 hours.

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Abstract

La présente invention concerne une arginase à mutation ponctuelle et son procédé de préparation, et l'utilisation de l'arginase à mutation ponctuelle dans la préparation de médicaments pour le traitement de maladies associées à l'arginase. La présente invention concerne également une arginase pégylée spécifique d'un site et son procédé de préparation, et l'utilisation de l'arginase pégylée dans la préparation de médicaments pour le traitement de maladies associées à l'arginase.
PCT/CN2012/087240 2011-12-27 2012-12-23 Arginase humaine et arginase humaine pégylée à point fixe, et leur utilisation Ceased WO2013097657A1 (fr)

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EP12861903.8A EP2799539B1 (fr) 2011-12-27 2012-12-23 Arginase humaine et arginase humaine pégylée à point fixe, et leur utilisation
US14/368,508 US20150010522A1 (en) 2011-12-27 2012-12-23 Human arginase and site-directed pegylated human arginase and the use thereof
BR112014015803-7A BR112014015803B1 (pt) 2011-12-27 2012-12-23 Arginase peguilada e composição farmacêutica para tratamento de uma doença relacionada com arginase
KR1020147020855A KR102076348B1 (ko) 2011-12-27 2012-12-23 사람 아르기나제 및 부위-지향성 페길화된 사람 아르기나제 및 그의 용도
JP2014549335A JP2015503333A (ja) 2011-12-27 2012-12-23 ヒトアルギナーゼおよび部位特異的ｐｅｇ化ヒトアルギナーゼとその使用方法

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JP2023522888A (ja) * 2020-04-17 2023-06-01 バイオ－キャンサー・トリートメント・インターナショナル・リミテッド アルギナーゼを使用してウイルス感染症を処置する方法
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US20240002535A1 (en) * 2020-11-30 2024-01-04 Merck Sharp & Dohme Llc Arginase 1 binders for inhibiting arginase 1 activity
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US9867875B2 (en) 2014-04-29 2018-01-16 Bio-Cancer Treatment International Limited Methods and compositions for modulating the immune system with Arginase I
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